Enhanced gene expression

ABSTRACT

The present disclosure is directed to a novel, unexpected approach of expressing exogenous gene(s) at increased levels by predictably, optionally irreversibly, incorporating the gene(s) into a region of increased gene expression (RIDGE) on a chromosome of a host cell. This approach is accomplished by identification of RIDGE(s) and further by integration of integrase-specific sites (e.g., attP or attB) in the presence of or mediated by integrase. The approach renders a high level of gene expression; predictability of the location of the exogenous genes, elimination of genetic instability or unwanted phenotype; and reduction of time and cost in optimizing protein production in host cells, which will be every useful in the production of therapeutic, prophylactic, and diagnostic proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 61/417,272, filed Nov. 25, 2010, which is specifically incorporatedby reference in its entirety.

FIELD OF THE INVENTION

The present disclosure relates to the field of gene expression.

BACKGROUND OF THE INVENTION

High level of gene expression in mammalian cells has always been achallenge to production of a large amount of proteins for diagnostic ortherapeutic uses. A typical process for protein production involves withthe steps of 1) transfecting a vector comprising a gene of interest intomammalian cells such that the vectors may randomly incorporate intosites of chromosomes of the cells through recombination and 2) screeningand selecting cells expressing a high level of expression of the gene ofinterest. Random recombination renders protein production as an art inscience, rather a predictable, repeatable scientific process.Additionally, the large amount of selection work is also a costly,burdensome, unpredictable process. Therefore, there is a great need inthe art to incorporate genes of interest into predictable, specificsites on chromosomes and reach a high level of gene expression.

SUMMARY OF THE INVENTION

One aspect of the present disclosure relates to methods of producingprotein at a high level comprising the steps of predictably placing anexogenous gene of interest into a region of increased gene expression(RIDGE) on a chromosome in a host cell and expressing the gene in thehost cell.

In certain embodiments, the methods herein include steps of identifyinga RIDGE on a chromosome in a host cell and introducing an anchor geneonto the RIDGE. The anchor gene can be a transgene of interest or anintegrase-specific site. The anchor gene can be introduced onto theRIDGE via homologous recombination.

In certain embodiments, the methods herein include steps of, forexample, identifying a RIDGE on a chromosome in a host cell andintroducing a first integrase-specific site to the RIDGE. The methodfurther comprise the steps of constructing a vector comprising a gene ofinterest and a second integrase-specific site and introducing the vectorinto the host cell in the presence of an integrase, wherein the gene isirreversibly incorporate into the chromosome and flanked by twointegrase-resulting sites. In certain embodiments, theintegrase-resulting sites are attL and attR.

Another aspect of the present disclosure relates to isolated nucleotidescomprising the sequence of at least a gene of interest and the sequenceof an integrase-specific site.

Another aspect of the present disclosure relates to a vector comprisingat least a gene of interest and an integrase-specific site.

Another aspect of the present disclosure relates to a host cellcomprising at least a gene of interest flanked by integrase-resultingsites (e.g., attL and attR).

Another aspect of the present disclosure relates to a host cell having aplurality of the same gene into a single RIDGE or a plurality of RIDGEs(e.g. a first RIDGE and a second RIDGE) respectively in a host cell.

Another aspect of the present disclosure relates to a host cell having afirst gene and a second gene into a single RIDGE or a first gene in afirst RIDGE and a second gene into a second RIDGE.

Another aspect of the present disclosure relates to method of producingproteins by expression a plurality of genes in the single host cellherein.

DRAWINGS

FIG. 1. A showing of how an integrase catalyzes irreversiblerecombination between attB and attP. For example, φC31 integrase is usedto mediate integration of a plasmid bearing a first integrase-specificsite (attP) into a chromosome containing a second integrase specificsite (attB), resulting an irreversible integration of the vector intothe chromosome and the vector is flanked by a set of integrase-resultingsites (attL and attR).

FIG. 2. A showing of how an attP site can be created in a RIDGE regionof a chromosome. The RIDGE region is screened or identified by randomintegration of a reporter cassette the GFP (Green Fluorescence Protein)and NEO (Neomycine resistant) genes flanked by two LoxP sites. 5′ to thecassette site contains an integrase (e.g., PhiC31 integrase) recognitionsite, attP, which will be integrated simultaneously with the cassette.The Cre/LoxP system is then used to remove the reporter cassette out ofthe RIDGE region and results in am attP site at the region.

FIG. 3. A showing of how a vector comprising a gene of interest (e.g., agene expressing an antibody or antigen-binding fragments thereof) and anattB site can be incorporated onto a chromosome in the presence of anintegrase.

FIG. 4. Examples of wide-typea ttB and attP sequences.

EMBODIMENTS OF THE INVENTION

One aspect of the present disclosure is directed to a novel, unexpectedapproach to express genes of interest at an increased level bypredictably, optionally irreversibly, incorporating the genes atspecific gene integration site(s) onto a chromosome of a host cell. Thepresent approach includes steps of, for example, identifying a specificgene integration site on a chromosome in a host cell; incorporating afirst integrase-specific (e.g., attP or attB) to the site, constructinga vector comprising a gene of interest and a second integrase-specific(e.g., attB or attP) site; incorporating the vector into the host cellin the presence of or mediated by an integrase, wherein the gene isirreversibly incorporate into the chromosome and flanked by attL andattR.

I. Identification of Specific Gene Integration Site(s).

The term “specific gene integration site” used herein refers to a siteor locus in a region of increased gene expression (RIDGE) on achromosome. The RIDGE is a region on a chromosome which genes residestherein tend to be highly expressed. When an exogenous gene isintroduced and incorporated onto a chromosome, the exogenous generesiding in a RIDGE tends to be expressed at a multiple folder higher(e.g., 2, 5, 10, 20, 50, 100, 200, 300, 500, 1000 folder higher) levelthan the same gene residing in a non-RIDGE or a randomly integratedsite.

RIDGEs have been genome-wide identified through transcriptome mappingwhere clusters of highly expressed genes reside therein. Table I showsthe RIDGEs that have been identified by comparative genomichybridization in brain, breast, liver and lung tissue cells (See, Zhouet al., Can.Res. 63:5781-5784 (2003)). See also Caron et al., Science291:1289-1292 (2001).

TABLE I (from Zhou et al., Can. Res. 63: 5781-5784 (2003) which isincorporated in its entirety by reference) CHR Brain Breast Liver Lung 11p34, 1p36, 1q21, 1q31 1p34, 1p33, 1q21, 1q41, 1q42 1p34, 1q21, 1q421p33, 1p34, 1p36, 1q21, 1q42 2 2p23, 2p11, 2q35, 2q36, 2q37 2p21, 2p24,2q11, 2q37 2p11, 2p25, 2q21, 2q35, 2q36 2p12, 2q11, 2q13, 2q21, 2q37 33p21, 3p22, 3q21, 3q28 3q25, 3q26, 3q29 3p21 3q21, 3q23, 3q27 4 4q13,4q21, 4q34 4p16 4p16 5 5p15, 5q35 5p15, 5q35 6 6p21, 6p24, 6q16 6p216p21 6p21 7 7p22, 7p12, 7p13, 7q22, 7q36 7q11, 7q22, 7q32, 7q34 7p13,7p12 7p22, 7p13, 7p12, 7q11, 7q22 8 8p21, 8q24 8q24 8q24 8q24 9 9p21,9q22, 9q34 9q32, 9q33 9p13, 9q34 9q22, 9q34 10 10q11, 10q22 10p15, 10q2610q22, 10q24 10p15 11 11p15, 11q12, 11q13 11q13, 11q12 11q12, 11q1311p15, 11p11, 11q12, 11q13 12 12p13, 12q13 12p11, 12q13, 12q24 12q1312p13, 12p11, 12q13, 12q24 13 13q14 14 14q32 14q11, 14q32 14q11, 14q3215 15q14, 15q15 15q14, 15q22, 15q23, 15q24 15q22, 15q24, 15q25, 15q26 1616p13, 16p12, 16p11, 16q12, 16p13, 16p12, 16p11, 16q24 16p13, 16p12,16p11, 16q22 16p13, 16p12, 16p11, 16q12, 16q24 16q22, 16q24 17 17q21,17q23, 17q25 17p13, 17p11, 17q21, 17q23, 17p13, 17q12, 17q21, 17q2517p11, 17q11, 17q21, 17q25 17q25 18 19 19p13, 19p12, 19q13 19p13, 19p12,19p11, 19q13 19p13, 19q13 19p13, 19p12, 19q13 20 20p13, 20q11, 20q1320q11, 20q13 20p13, 20q11 20p13, 20q11, 20q13 21 21q22 21q21 21q22 2222q12, 22q13 22q12, 22q13 22q13 22q11, 22q12 X Xp11, Xq11, Xq12, Xq13,Xq28 Xp22, Xq12, Xq13, Xq22, Xq28 Xp11, Xq28

RIDGEs can also be identified through systems and methods as disclosedin Jiao et al. titled “retargeting of pre-set regions on chromosome forhigh gene expression in mammalian cells.” (Seehttp://hdl.handle.net/1721.1/7491 (2005)). Briefly, a vector containinggfp2 reporter gene was transfected into mammalian cells (e.g., Chinesehamster ovary (CHO) cells) and the cells with highest expression levelof GFP were selected where the linearized vector was integrated into thechromosome of the CHO cells and resided in a RIDGE. A gene of interest,for example, Interferon, was then used to replace gfp2 gene and the CHOcells with dimmest GFP fluorescence were selected with the ability ofhighly expressing interferon.

RIDGEs can also be identified by using the microarray analysis of wholecell or cytoplasm mRNA transcription level and identifies those ofhigher transcription (e.g., (e.g., 2, 5, 10, 20, 50, 100, 200, 300, 500,1000 folder higher) than house-keeping mRNAs like beta-actin andidentifying corresponding exon regions transcribing those RNA.

The specific gene integration site includes at least one of thefollowing characteristics: 1) insertion of an exogenous gene should notdisrupt the functions of regulatory elements or genes in the RIDGE orchromosome; 2) the site should be in an intergenic region; 3) the siteshould be spatially and temporally ubiquitous active; 4) the site shouldbe transcriptional active at chromosomal level such that thetranscription machinery is active at the site and a higher level oftranscription; and 5) insertion of the exogenous gene should notinterference the viability of the host cell.

The specific gene integration site contains nucleotide sequences knownin the art. For example, DNA marker sequences of the RIDGEs in Table Iare well known and readily accessible through gene data bank.

In certain embodiment, once the RIDGEs and specific gene integrationsites are identified, transgenes can be incorporated into the sitethrough recombination. For example, conventional targeting vectors canbe engineered for insertion of transgenes at selected sites in thegenome of interest where the vectors consist of a 5′ homology arm (tothe 3′ of the site), followed by the transgene of interest (frequentlypreceded by a particular promoter or promoter-less), a positiveselection marker gene-containing cassette, and a 3′ homology arm (to the5′ of the site). The selection marker gene-containing cassette used inthese methods consists of a ubiquitously expressed promoter such as thephosphoglycerate kinase promoter which drives the expression of apositive drug selection gene such as neomycin phosphotransferase orother suitable drug selection gene familiar in the art, followed by apolyadenylation signal sequence to confer efficient polyadenylation ofthe transcribed message. The selection cassette confers drug resistancewhen the vector integrates at the desired specific gene integration sitevia homologous recombination. The transcription or expression levels oftransgenes are then analyzed.

In certain embodiment, integrase-specific sites are incorporated intothe RIDGEs and specific gene integration sites via homologousrecombination such that cells embodying the integrase-specific site(s)can be used as mater cell lines for incorporating different genes ofinterest when desired.

II. Incorporation of at Least a First Integrase-Specific Site to atLeast a Specific Gene Integration Site

The term “integrase-specific site” or “ISS” used herein means attP orattB. When a first ISS is attP, a corresponding second ISS is attB (orthe first ISS is attB and the second ISS is attP) such that the firstISS and the second ISS can undergo site specific integration mediated byor in presence of an integrase.

In certain embodiments, RIDGEs are identified in accordance with thedisclosed methods in Jiao et al. titled “retargeting of pre-set regionson chromosome for high gene expression in mammalian cells.” (Seehttp://hdl.handle.net/1721.1/7491 (2005)) and the report gene gfp2 wasincorporated into a RIDGE in the CHO cells.

As shown in FIG. 2, the gene sequence of gfp2 (or a fraction thereof),namely “U”, can be used as a base for homologous recombination. A vectorcomprising the sequence of “U” as well as attP and double selectiongenes (e.g., Neo or hrGFP) flanked by loxP is introduced to the CHOcells and attP was incorporated adjacent to “U” along with Neo-hrGFP.The Neo & hRGFP can be reversibly removed by Cre enzyme. As a result,full attP is incorporated adjacently to the specific gene integrationsite and in the RIDGE.

In certain embodiments, RIDGEs have been genome-wide identified throughmethods disclosed by Zhou et al., Can.Res. 63:5781-5784 (2003). Aspecific DNA marker sequence in, for example, Chromosome II 11q13, knownin the art, is used as an anchor sequence “U” as shown in FIG. 2.

In certain embodiments, Chromosome 11 ROSA 26 locus has been identifiedas a transcription active locus which is accessible to the transcriptionmachinery and RNAs resulting from the transcription can be found insidecells (See U.S. Pat. No. 7,473,557). Sequences downstream of exon 1 ofthe ROSA26 locus can be used as an anchor sequence “U” as shown in FIG.2.

In certain embodiments, there are a plurality “U”s or a plurality ofspecific gene integration sites (each with same or different sequences)along a chromosome or in different chromosomes so as to render aplurality of integrase-specific sites incorporated into a chromosome orchromosomes.

III. Construction of a Vector Comprising at Least a Gene of Interest anda Second Integrase-Specific Site

As known in the art, a vector comprising at least a gene of interest anda second integrase-specific site can be readily constructed. The secondintergrase-specific site is a corresponding site to the first ISS wherethe first and second will engage in a site specific integration in thepresence of an integrase (See FIG. 1). As shown in FIG. 3, a targetingvector is constructed to contain a gene of interest, an attB site, and amarket gene DHFR.

The gene of interest can be a gene encoding a protein of interest fortherapeutic, diagnostic, or prophylactic purposes. For example, aprotein of interest can be any one or more of the following antigensincluding but not limited to:

-   -   leukocyte markers, such as CD2, CD3, CD4, CD5, CD6, CD7, CD8,        CD11a,b,c, CD13, CD14, CD18, CD19, CD20, CD22, CD23, CD27 and        its ligand, CD28 and its ligands B7.1, B7.2, B7.3, CD29 and its        ligand, CD30 and its ligand, CD40 and its ligand gp39, CD44,        CD45 and isoforms, CDw52 (Campath antigen), CD56, CD58, CD69,        CD72, CTLA-4, LFA-1 and TCR;    -   histocompatibility antigens, such as MHC class I or II, the        Lewis Y antigens, SLex, SLey, SLea, and SLeb;    -   integrins, such as VLA-1, VLA-2, VLA-3, VLA-4, VLA-5, VLA-6, and        LFA-1;    -   adhesion molecules, such as Mac-1 and p150,95;    -   selectins, such as L-selectin, P-selectin, and E-selectin and        their counterreceptors VCAM-1, ICAM-1, ICAM-2, and LFA-3;    -   interleukins, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,        IL-8, IL-10, IL-11, IL-12, IL-13, IL-14; and IL-15;    -   interleukin receptors, such as IL-1R, 1L-2R, IL-4R, IL-5R,        IL-6R, IL-7R, IL-8R, IL-10R, IL-11R, IL-12R, IL-13R, IL-14R, and        IL-15R;    -   chemokines, such as PF4, RANTES, MIP1.alpha., MCP1, NAP-2, Grou,        Grog, and IL-8;    -   growth factors, such as TNFalpha, TGFbeta, TSH, VEGF/VPF, PTHrP,        EGF family, FGF, PDGF family, endothelin, and gastrin releasing        peptide (GRP);    -   growth factor receptors, such as TNFalphaR, RGFbetaR, TSHR,        VEGFR/VPFR, FGFR, EGFR, PTHrPR, PDGFR family, EPO-R, GCSF-R and        other hematopoietic receptors;    -   interferon receptors, such as IFN.alpha.R, IFN.beta.R, and        IFN.gamma.R;    -   Igs and their receptors, such as IgE, FceRI, and FCeRII;    -   tumor antigens, such as her2-neu, mucin, CEA and endosialin;    -   allergens, such as house dust mite antigen, IoI p1 (grass)        antigens, and urushiol;    -   viral proteins, such as CMV glycoproteins B, H, and gCII, HIV-1        envelope glycoproteins, RSV envelope glycoproteins, HSV envelope        glycoproteins, EBV envelope glycoproteins, VZV envelope        glycoproteins, HPV envelope glycoproteins, Hepatitis family        surface antigens;    -   toxins, such as pseudomonas endotoxin and osteopontin/uropontin,        snake venom, and bee venom;    -   blood factors, such as complement C3b, complement C5a,        complement C5b-9, Rh factor, fibrinogen, fibrin, and myelin        associated growth inhibitor;    -   enzymes, such as cholesterol ester transfer protein, membrane        bound matrix metalloproteases, and glutamic acid decarboxylase        (GAD); and    -   miscellaneous antigens including ganglioside GD3, ganglioside        GM2, LMP1, LMP2, eosinophil major basic protein, eosinophil        cationic protein, PANCA, Amadori protein, Type IV collagen,        glycated lipids, .gamma.-interferon, A7, P-glycoprotein and Fas        (AFO-1) and oxidized-LDL.

In certain embodiments, the protein of interest can be antibodies orfragments thereof which bind to an antigen (non-limiting example ofantigens are shown as above). The antibodies or fragments can bepolyclonal, monoclonal, of animal origin (e.g., murine, rabbit, camel),of human origin (e.g., fully human), chimeric, humanized, variableregions, CDRs, ScFv, bispecific, diabody, or other forms of antibodieswith antigen-binding capabilities.

In certain embodiment, the vector can contain a plurality of genes ofinterest. Vectors herein include palsmids which are capable ofexpressing DNA sequences contained therein, where such sequences areoperably linked to other sequences capable of effecting theirexpression, i.e., promotor/operator sequences. A vector is given afunctional definition: any DNA sequence which is capable of effectingexpression of a specified DNA code disposed therein.

IV. Incorporation of a Vector Comprising at Least a Gene of Interest andan Integrase-Specific Site to a Chromosome Comprising at Least AnotherIntegrase-Specific Site in Presence Of or Mediated by an Integrase.

As shown in FIG. 3, the vector containing a gene of interest and attB isintroduced to a host cell having a chromosome wherein an attP sited isintegrated as disclosed above. In the presence of or mediated by anintegrase (e.g., φC31 integrase) in the host cell, the attP isintegrated with attB so as to integrate the gene of interest into thechromosome. As a result, the gene of interest is flanked by twointegrase-resulting sites (attL and attR) and located in or adjacent toa specific gene integration site in the region of increased geneexpression (RIDGE) on a chromosome in a host cell.

In certain embodiments, the integrase is a resolvase or invertase or amember of the serine recombinase family of site-specific recombinases.For example, the integrase is φC31(gene ID: 2715866); R4 (gene ID:1099373), an integrase from the Streptomyces phagesTP901-1(gene ID:921049 and 921048), an integrase from the lactococcal phage SpoIVCA(gene ID: 937799), a recombinase that excises a prophage-like elementfrom the Bacillus genome during sporulation.

In certain embodiments, host cells herein are cells which are capable ofbeing transformed by a vector. Host cells can be prokaryotic (e.g.,bacteria) or eukaryotic (e.g., yeast, plant, mammalian cells like CHOcells).

In certain embodiments, an integrase is present in a host cell byintroducing an integrase gene-containing vector (including promoters andother elements for expression) into the host cell so that integrase canexpressed in the cell.

In certain embodiments, an integrase-specific site can be attB (SEQ IDNO. 1) or homologies of attB (e.g., 98%, 95%, 90%, 85%, 80% homologousto SEQ ID NO. 1) or attP (SEQ ID NO. 2) or homologies of attP (e.g.,98%, 95%, 90%, 85%, 80% homologous to SEQ ID NO. 2). FIG. 4 shows anexample of wide-type attB and attP sequences.

In certain embodiments, an integrase-resulting site is the sequenceresulting from the integration of two corresponding integrase-specificsites. For example, an integrase-resulting site is attL or attR. attLcan have a nucleotide sequence of GTGCCAGGGCGTGCCCTTGAGTTCTCAGTTGGGGG(SEQ ID NO. 3) or a homology of thereof (e.g., 98%, 95%, 90%, 85%, 80%homologous to SEQ ID NO. 3). attR can have a nucleotide sequence ofCCCCAACTGGGGTAACCTTTGGGCTCCCCGGGCGCG (SEQ ID NO. 4) or a homologythereof (e.g., 98%, 95%, 90%, 85%, 80% homologous to SEQ ID NO. 4).

In certain embodiments, a plurality of vectors comprising a plurality ofgenes can be integrated into a plurality of integrase-specific sites ona chromosome or a plurality of chromosomes in a single host cell.

In certain embodiments, a gene of interest is a gene expressing anantibody or a fragment thereof. After integration into the RIDGE on achromosome, the expression level of the antibody is multiple fold (e.g.,2, 3, 4, 5, 10, 20, 50, 100) higher than the level of randomintegration. For example, the antibody expression level reaches 1pg/cell/day, 2 pg/cell/day, 5 pg/cell/day, 10 pg/cell/day, 20pg/cell/day, 50 pg/cell/day, or 100 pg/cell/day.

Advantages. The present disclosure renders a great benefit to theprotein production. The present approach renders a high level of geneexpression by integrating exogenous genes into region(s) of increasedgene expression (RIDGE) on a chromosome of a host cell and ensuring arobust gene expression. The approach disclosed herein providespredictability of the location of the exogenous genes, eliminatesgenetic instability or unwanted phenotype caused by multiple rounds ofamplifications and screening; and reduces the time and cost inoptimizing protein production in host cells, which will be every usefulin the production of therapeutic, prophylactic, and diagnostic proteins.In addition, after a first integrase-specific site is introduced into aRIDGE in a host cell, the host cell can be used as a master cell linefor expression of various genes of interest, since a gene of interestcan be easily incorporated onto the first site by introducing theintegrase-present host cell with a vector containing the gene ofinterest and a second integrase-specific site.

1. A host cell comprising a first integrase-specific site, wherein thesite is located at a region of increased gene expression (RIDGE) on achromosome in the host cell.
 2. The host cell of claim 1 wherein thefirst integrase-specific site is attP or attB or a homology thereof. 3.The host cell of claim 1 further comprising a vector, wherein the vectorcomprising an exogenous gene and a second integrase-specific site andthe second site is capable of being integrated with the first site. 4.The host cell of claim 3 further comprising an integrase.
 5. A host cellcomprising an exogenous gene flanked by a first integrase-resulting siteand a second integrase-resulting site, wherein the gene is located at aregion of increased gene expression (RIDGE) on a chromosome of the cell.6. The host cell of claim 5 wherein the first integrase-resulting siteis attL or a homology thereof.
 7. The host cell of claim 5 wherein thesecond integrase-resulting site is attR or a homology thereof.
 8. Amethod of expressing a gene comprising a step of introducing a firstintegrase-specific site to a region of increased gene expression.
 9. Themethod of claim 8 further comprising a step of constructing a vectorcomprising an exogenous gene and a second integrase-specific site. 10.The method of claim 9 further comprising a step of introducing thevector into the host cell in the presence of or mediated by anintegrase, wherein the gene is incorporated into the chromosome andflanked by a first integrase-resulting site and a secondintegrase-resulting site.
 11. A vector comprising a first gene, a secondgene, and an integrase-specific site.
 12. A host cell comprising a firstintegrase-specific site and a second integrase-specific site, whereinboth sites are introduced into the host cell.
 13. The host cell of claim12 where in the first and second sites are in a single region ofincreased gene expression.
 14. The host cell of claim 12 wherein thefirst site is in a first region of increased gene expression and thesecond site is in a second region of increased gene expression.
 15. Ahost cell comprising a first exogenous gene and a second exogenous gene,wherein the first gene is flanked by a set of first integrase-resultingsites and the second gene are flanked by a set of secondintegrase-resulting sites.
 16. The host cell of claim 15 wherein the setof the first integrase-resulting sites is the same or not the same asthe set of the second integrase-resulting sites.
 17. The host cell ofclaim 15 wherein the first and second genes are in a single region ofincreased gene expression.
 18. The host cell of claim 15 wherein thefirst gene is in a first region of increased gene expression and thesecond gene is in a second region of increased gene expression.
 19. Thehost cell of claim 18 wherein the first and second regions are in asingle chromosome.
 20. The host cell of claim 18 wherein the firstregion is in a first chromosome and the second region is in a secondchromosome.
 21. A method of highly expression a gene of interest in ahost cell, comprising the steps of: a) identifying at least one regionof increased gene expression (RIDGE) on a chromosome in the host cell;b) introducing an anchor gene into the RIDGE.
 22. The method of claim 21where in the anchor gene is a transgene of interest, wherein thetransgene encodes a protein of interest.
 23. The method of claim 21where in the anchor gene is a first integrase-specific site.
 24. Themethod of claim 23 further comprising a step of introducing the vectorinto the host cell in the presence of or mediated by an integrase,wherein the vector comprising an exogenous gene and a secondintegrase-specific site, and the exogenous gene is incorporated into thechromosome and flanked by a first integrase-resulting site and a secondintegrase-resulting site.